Directed Therapy to Combat Mycobacterial Infections*

Received: 18 December 2020 | Accepted: 27 December 2020 DOI: 10.1111/imr.12951

INVITED REVIEW

Host- directed therapy to combat mycobacterial infections*

Gül Kilinç | Anno Saris | Tom H. M. Ottenhoff | Mariëlle C. Haks

Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Abstract Netherlands Upon infection, mycobacteria, such as Mycobacterium tuberculosis (Mtb) and nontu-

Correspondence berculous mycobacteria (NTM), are recognized by host innate immune cells, triggering Mariëlle C. Haks, Department of a series of intracellular processes that promote mycobacterial killing. Mycobacteria, Infectious Diseases, Leiden University Medical Center, Albinusdreef 2, ZA Leiden however, have developed multiple counter- strategies to persist and survive inside host 2333, The Netherlands. cells. By manipulating host effector mechanisms, including phagosome maturation, Email: [email protected] vacuolar escape, autophagy, antigen presentation, and metabolic pathways, patho- Funding information genic mycobacteria are able to establish long-lasting infection. Counteracting these Netherlands Organisation for Scientific Research (NWO- TTW), Grant/Award mycobacteria- induced host modifying mechanisms can be accomplished by host- Number: 16444; European Union (Horizon directed therapeutic (HDT) strategies. HDTs offer several major advantages compared 2020 programme), Grant/Award Number: 847762; IMI2 JU (AMR Accelerator to conventional antibiotics: (a) HDTs can be effective against both drug- resistant and program), Grant/Award Number: 853932 drug- susceptible bacteria, as well as potentially dormant mycobacteria; (b) HDTs are and 853903 less likely to induce bacterial drug resistance; and (c) HDTs could synergize with, or shorten antibiotic treatment by targeting different pathways. In this review, we will explore host- pathogen interactions that have been identified for Mtb for which potential HDTs impacting both innate and adaptive immunity are available, and outline those worthy of future research. We will also discuss possibilities to target NTM infection by HDT, although current knowledge regarding host- pathogen interactions for NTM is limited compared to Mtb. Finally, we speculate that combinatorial HDT strategies can potentially synergize to achieve optimal mycobacterial host immune control.

KEYWORDS drug resistance, host- directed therapy, Mycobacterium avium, Mycobacterium tuberculosis, nontuberculous mycobacteria

1 | INTRODUCTION of deaths globally.1 Approximately a quarter of the world's population is infected with Mtb and in most cases, progression toward TB Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), re- disease is prevented by an efficient host immune response, often mains a major health problem. With an estimated 10 million disease resulting in a latent TB infection (LTBI).1 Five to fifteen percent of cases and 1.4 million deaths in 2019, Mtb is the deadliest infec- LTBI individuals will develop TB disease during their life- time, often tious agent worldwide and TB is one of the top- 10 leading causes concomitant with host immunocompromising conditions, including

This article is part of a series of reviews covering Immunity to Mycobacteria appearing in HIV infection and use of immunosuppressive medication. Treatment Volume 301 of Immunological Reviews. of patients with active TB has largely remained unchanged for over Kilinç, Saris, Ottenhoff, and Haks authors are Contributed equally. 30 years,1 and due to its lengthiness (6- 24 months) and considerable

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. © 2021 The Authors. Immunological Reviews published by John Wiley & Sons Ltd.

Immunological Reviews. 2021;00:1–22. wileyonlinelibrary.com/journal/imr | 1 2 | KILINÇ et aL side effects, treatment- adherence is low fueling development of regimens, thereby increasing compliance. Fifth, HDT may permit multidrug and extensive- drug resistance (MDR and XDR). The large dose lowering of standard antibiotics, thus reducing toxicity with- TB disease burden and the increasing incidence of drug resistance out impacting efficacy. Finally, as HDT and mycobacterium- targeting make alternative treatment solutions imperative. compounds (ie, antibiotics) by definition act on different pathways, While the number of TB cases is slowly declining, a trend that combinatorial regimens would be expected to synergize. In this re- may well be broken as a result of the COVID- 19 pandemic,2 the prev- view, we will provide a comprehensive overview of host- pathogen alence of infections known to be caused by nontuberculous myco- interactions that have been identified in Mtb infections and that are bacteria (NTM) is increasing at an alarming rate, currently reaching amenable to targeting by HDTs (summarized in Figure 1 and Table 1). 0.2- 9.8 per 100.000 individuals.3 NTM represent a group of op- Furthermore, despite a limited number of reports, we will also dis- portunistic mycobacterial pathogens that mostly cause pulmonary cuss NTM- mediated host modulation and speculate whether HDTs diseases (PD), predominantly in vulnerable populations due to immu- could also be of interest to combat these mycobacterial infections. nodeficiencies and/or pre- existing lung conditions. Mycobacterium Finally, we will discuss the possibility of combinatorial HDTs that tar- avium (Mav) complex (MAC) and Mycobacterium abscessus (Mab) ac- get distinct host signaling pathways to promote possible synergistic count for the large majority of reported cases.3 Despite extended treatment effects. treatment regimens, clinical outcome is poor, with cure rates of approximately 50%- 88% among MAC- PD patients and 25%- 58% among Mab- infected individuals,3 urging the development of novel 2 | HDT MODULATING INNATE IMMUNE treatment modalities. CELL FUNCTION Mycobacteria are well known for their capability to manipulate intracellular signaling pathways to escape from host- defense mech- 2.1 | Phagocytosis and phagosome maturation anisms in human cells. Mtb is best studied in this regard, but NTM have also been shown to modulate host immune responses, includ- The first potential target for HDT to interfere with host- pathogen in- ing preventing phagosome acidification and maturation or escap- teractions is to modulate mycobacterial host- cell entry. Mycobacteria ing from phagosomes into the nutrient- rich cytosol. Counteracting infect host cells, predominantly alveolar macrophages and epithe- pathogen- induced immune modulation by host- directed therapy lial cells, in the lower respiratory tract, following inhalation of small (HDT) is a promising adjunct therapy to antibiotic therapy to combat bacteria- containing aerosols. Mycobacteria express pathogen- intracellular mycobacterial infections, with several major advantages associated molecular patterns (PAMPs) that are recognized by pat- over current antibiotics. First, HDT can also be effective against tern recognition receptors (PRRs), such as Toll- like receptors (TLRs), MDR/XDR mycobacteria that are insensitive to current standard C- type lectin receptors, and other scavenger receptors expressed on antibiotics. Second, because there is no direct selection pressure on the surface of host cells to initiate phagocytosis.4 Especially TLR2, mycobacteria, host- targeting compounds are less likely to result in which forms heterodimers with TLR1 and TLR6 to recognize lipo- drug resistance. Third, host- targeting compounds have the potential proteins or lipopeptides (eg, lipomannan), and TLR4, which recog- to target metabolically inactive, non- replicating bacilli during LTBI, nizes cell wall lipids, glycoproteins, and secreted proteins, are known which are tolerant or resistant to conventional therapies. Fourth, to mediate Mtb- induced cellular activation.5 Mice lacking TLR2 are HDT may allow shortening of current lengthy TB/NTM- treatment more susceptible to infections with virulent Mtb and Mav strains.6- 8

FIGURE 1 Host- pathogen interactions and potential host- directed therapies (HDT). Granulomas are characteristic for tuberculosis and mycobacterial infections in general. Pathologic granulomas are poorly vascularized due to ineffective angiogenesis, leading to hypoxia and concomitant host- cell necrosis and bacterial dissemination. Blocking angiogenesis, preventing host- cell necrosis (or stimulating apoptosis) or inhibiting extracellular matrix (ECM) degradation improves granuloma structure and concomitant disease outcome. Macrophages, key cells in the antimycobacterial response, initiate phagocytosis after toll- like receptor (TLR) recognition, which is prevented and/or modulated by mycobacteria. Promoting TLR4 engagement, TLR2 signaling and post- phagocytic signaling via receptor tyrosine kinase are all potential targets for HDT to improve host immunity during mycobacterial infection. After internalization, mycobacteria are located to phagosomes that slowly mature and ultimately fuse with lysosomes, which are all inhibited by mycobacteria. Alternatively, mycobacteria escape to the cytosol where they can be recognized by cytoplasmic pathogen recognition receptor (PRR) and “recaptured” using autophagy, which again is inhibited by mycobacteria. HDTs that (1) prevent phagosomal escape, (2) alleviate blockage of (auto- )phagosome maturation, (3) promote autophagy and/or (4) stimulate (auto- )phagolysosome fusion all enhance mycobacterial killing. HDT that enhance cytoplasmic recognition of mycobacteria also improve the antimycobacterial immune response. Mycobacteria that remain in the cytosol impair host metabolic pathways by stimulating tricarboxylic acid (TCA) cycle intermediates from mitochondria to be expelled into the cytosol to form lipid droplets and induce mitochondrial membrane depolarization. HDTs that (1) impair lipid droplet accumulation, (2) prevent mitochondrial membrane depolarization, and/or (3) stimulate TCA cycle intermediates being allocated in eicosanoid signaling maintain macrophage functionality which leads to better mycobacterial control. Finally, mycobacteria prevent the host from mounting an effective adaptive immune response by inhibiting antigen presentation and impairing T- cell skewing. HDTs that promote adaptive immunity by enhancing antigen presentation, stimulating Th1 skewing or inhibiting Th2/Treg immunity all improve disease outcome. Compounds that can correct the above processes are represented in red for inhibitory/blocking therapies and in green for stimulatory therapies and summarized in Table 1, ordered per physiological process KILINÇ et aL | 3

Inhibit MMP and/or ECM degradation

Stimulate Th1 immunity Block angiogenesis (and mediated signalling)

Granuloma

Angiogenesis

Host-cell death

Prevent host-cell necrosis and ferroptosis or stimulate apoptosis

Phagocytosis Promote TLR2 and Promote TLR4 engagement post-phagocytic signaling

Enhance cytoplasmic pathogen recognition

Stimulate host-protective epigentics N N

F L cytokines

- κ

P B

3 Intracytoplasmic pathogen Restore immune balance by: Stimulate phagosome maturation recognition Eicanosoid limiting immunity via IL-1/prostaglandin signaling signaling Phagosome IL-1 or promoting immunity via type I IFN/leukotriene signaling maturation family

Phagosomal Leukotrienes Prostaglandins escape Type I IFN TCA Promote autophagy intermediates Autophagy/ Inhibit Th2 immunity Xenophagy Metabolism Inhibit lipid droplet accumulation Stimulate Th1 immunity (Auto-)phago Phagosomal lysosome fusion escape

RIG-I-like / NOD Stimulatory HDT receptors Prevent Co-stimulatory Inhibitory HDT phagosomal escape ligand Mycobacteria- Co-stimulatory mediated inhibition receptor Prevent mitochondrial Macrophage dysfunction TLR4 Antigen TLR2 Th1/Th1*/Th17 T cell T cell activation presentation TCR Monocyte and skewing

MHC class II/I B cell proton pump Dendritic cell IFN-y IL-17 Neutrophil IL-23 Lysosome IL-1β Lipid droplet IL-4 Enhance antigen TCA cycle presentation IL-24 Mitochondria with IL-12 functional OXPHOS Stimulate (auto-)phago- Depolarized lysosome fusion ECM mitochondria

NTM, including Mav and Mab, express a class of glycolipids known TLR2- MyD88 pathway has been linked to phagolysosome escape of as glycopeptidolipids (GPLs) which mask underlying cell wall virulent Mtb to the cytosol,11,12 indicating a crucial role for TLR sign- phosphatidyl- myo- inositol mannosides, thereby limiting interactions aling pathways in the control of intracellular mycobacteria. Several with TLR2.9,10 Moreover, the acetylation state of lipomannan modu- PRR agonists, including TLR2 agonist Pam2Cys, have been identified lates TLR2- mediated macrophage activation, and subversion of the that activate both innate and adaptive immune responses against 4 | KILINÇ et aL 140 (Continues) 160 149 160,188,191 121 116 122 13 124 129 133,134,137- 141 142 145 131 146- 151 152 158- 155 99 161 161 157- Ref. Mtb Mtb Mtb Mtb Mtb Mtb Mtb Mtb, Mav Mtb Mtb Mtb Mtb Mtb, Mav Mtb Mtb Mtb Mtb Mtb Mtb Pathogen Mtb in vitro ex vivo, in vivo ex vivo, in vivo in vitro ex vivo, in vivo in vitro, in vivo in vitro, in vivo in vitro, (R)CT in vivo in vivo in vivo in vitro in vivo, (R)CT in vivo CR (R)CT in vitro in vitro in vitro, in vivo, R(CT) Model in vitro α α signaling) signaling) ↑ Th1 immunity↑ Th1 ↑ Th1 immunity↑ Th1 ↑ Antigen presentation ↑ Antigen presentation immunity↑ Th1 (TLR4 immunity↑ Th1 (TLR4 immunity↑ Th1 immunity↑ Th1 immunity↑ Th1 ↓ Immune response ↓ Th2 immunity immunity↑ Th1 immunity↑ Th1 immunity↑ Th1 ↓ Immune response ↑ Apoptosis ↓ Necrosis ↑ TNF- ↓ Necrosis ↓ Immune response Biological activity ↑ TNF- Cells 4 cytokines

+ 106binhib. a

CD40 agonist HMBPP

ZM1 γ 1 inhib. + +

89 1MT 24 2 2 12 Bergenin MiRNA- AGK2 G1- LPS D- Skewing of T- IFN- IL- IL- FPS- Blocking IL- IL- IL- PBMCs Prednisolone (PROGRAMMED) cell death Mcl- Nicotinamide SQ22536 Dexamethasone Compound Antigen presentation and priming H- 186 224 47,51,52 92 17 77 13- 19 20 26,27 28 29 29 29 33,34 71- 78 84,222- 85 86- 70 70 187 181,184- 26,27 40- Ref. Mtb Mtb Mtb Mtb, Mm Mtb Mtb Mtb Mtb Mtb, Mav Mtb Mtb Mtb Mtb Pathogen Mtb Mtb Mtb Mtb Mtb Mtb Mtb (R)CT (R)CT (R)CT CR (R)CT in vitro, in vivo, in vitro, in vivo, in vitro in vitro, in vivo, in vitro in vivo, (R)CT in vitro in vitro, in vivo in vitro, in vivo, in vitro, in vivo in vitro in vitro in vitro, in vivo Model in vitro in vivo in vitro in vitro in vitro, (R)CT in vitro, (R)CT in vitro, in vivo, phagolysosome phagolysosome phagolysosome phagolysosome phagolysosome fusion fusion fusion fusion fusion recognition ↑ Phagosome maturation ↑ (Auto)- ↑ TLR2 signaling ↑ Phagosomal acidification ↑ Autophagy ↑ Phagocytosis ↓ Phagocytosis Unknown ↑ (Auto)- ↑ Phagocytosis ↑ (Auto)- ↑ Lysosomal biogenesis ↑ (Auto)- ↑ TLR2 signaling ↑ (auto)- Biological activity ↑ PRR signaling ↑ Phagosomal acidification Unknown Unknown ↑ Cytoplasmic pathogen ↑ Autophagy ↑ Phagosome maturation

5p inhib.

a a

HDT compounds HDT and their biological activity against mycobacterial infections

a a D a a 23a- 125 inhib.

2076 a

a CSF ENMD- SRT1720 Compound Phagocytosis and phagosome maturation PRR agonists miRNA- Imatinib AZD0530 AT9283 Dovitinib Nitazoxanide Metformin Gefitinib Bedaquiline 2062 GM- Resveratrol Celecoxib Statins miRNA- Autophagy Imatinib Vitamin- TABLE 1 KILINÇ et aL | 5 186 (Continues) 157 163 163 164,165 164,165 171 129 176,177 174 175 176,177 180 179 181,184- 189,190 187 187 192,193 Ref. Mm Mtb Mtb Mtb, Mav Mtb Mtb Mm Mtb Mm Mtb Mtb Mtb Mtb Mtb Mtb Mtb Mtb, Mav Pathogen Mtb Mtb in vivo in vivo in vivo in vitro in vitro, in vivo in vitro in vivo in vitro in vivo in vitro in vitro in vivo in vivo, (R)CT in vivo ex vivo, in vivo in vivo, (R)CT in vitro Model in vitro, in vivo, (R)CT in vivo α α α /prostaglandin /prostaglandin /prostaglandin /prostaglandin β β β β 1 1 1 1 dysfunction accumulation signaling signaling signaling signaling incorporation ↑ Apoptosis ↓ Ferroptosis ↓ Necrosis ↓ TNF- ↓ Necrosis ↓ Necrosis ↓ TNF- ↓ TNF- ↓ Glycolysis ↑ Immune response? ↓ Mitochondrial ↓ Lipid droplet ↑ IL- ↓ IL- ↑ IL- ↑ Apoptosis ↓ IL- ↑ Iron chelation Biological activity ↓ Membranal cholesterol ↓ Glycolysis

a a

a a 4 Zileuton 1MT DG Ferrostatin 1 Alisporivir Doramapimod Desipramine Cilostazol Sildenafil D- administrationATP Carbohydrate and lipids 2- administrationATP Clemastine M1 Ezetimibe Statins Eicosanoids PGE2 and/or Celecoxib LTB Ibuprofen Compound Antigen presentation and priming FX11 186 224 109 62 53 101 77 55- 49- 66 68 69 71- 78 79 80 84,222- 70 70 19 20 181,184- 95,97 99- 107- 79 Ref. Mtb Mtb Mtb Mtb Mtb Mtb Mtb Mtb Mtb Mtb Mtb Mtb Mtb Mtb Pathogen Mtb Mtb Mtb Mtb Mtb Mtb (R)CT (R)CT (R)CT (R)CT (R)CT in vivo in vitro, in vivo, in vitro, in vivo, in vitro, in vivo, in vitro, in vivo, in vitro in vitro in vitro in vitro in vitro, in vivo in vitro, in vivo in vitro in vitro, in vivo in vitro Model in vitro, (R)CT in vitro, (R)CT in vitro, ex vivo in vitro, ex vivo (R)CT in vitro, in vivo, dysfunction ↑ Autophagy ↓ Reactive oxygen species ↑ Autophagy, ↑ Autophagy ↑ Autophagy ↑ Autophagy ↑ Reactive oxygen species ↑ Autophagy ↓ Mitochondrial ↑ Reactive oxygen species ↑ Autophagy ↑ Autophagy ↑ Autophagy ↑ Autophagy ↑ Autophagy Biological activity ↑ Autophagy ↑ Autophagy ↑ Autophagy ↑ Autophagy ↑ Reactive nitrogen species ↑ Autophagy

a ) 2+

a a

5p inhib.

a a (Continued)

cysteine a a A (± Zn 27a antagomir 27a 23a- 125 inhib.

a acetyl- arginine Bedaquiline CD157 Vitamin- Compound Phagocytosis and phagosome maturation PBA MiRNA- Everolimus Ibrutinib Metformin Gefitinib Bazedoxifene Loperamide 2062 Resveratrol SRT1720 miRNA- Statins Intracellular killing mechanisms N- L- Bazedoxifene miRNA- TABLE 1 6 | KILINÇ et aL DG, 198 ; (R)CT,; 193- 208 25 212,213 166 210,214 203,205 205 205 217 218 218 219 Ref. Mtb Mtb Mm Mtb Mtb Mtb Mtb Mtb Mtb Mtb Mm Mm Mm Pathogen Mm, Mycobacterium marinum ; in vivo, (R)CT in vivo in vivo in vitro, in vivo in vivo in vitro, in vivo, (R)CT in vitro, in vivo in vivo in vivo in vivo in vivo in vivo in vivo Model stimulating factor; PBA, phenylbutyrate; SAHA, suberoylanilide Mav, Mycobacterium avium ; /prostaglandin β 1 signaling enyl pyrophosphate; PBMCs, peripheral blood mononuclear cells; 2- ↓ IL- ↓ MMP/ECM degradation↓ MMP/ECM degradation↓ MMP/ECM degradation↓ MMP/ECM degradation↓ MMP/ECM degradation↓ MMP/ECM degradation↓ MMP/ECM degradation↓ MMP/ECM degradation↓ MMP/ECM ↓ Angiogenesis ↓ Angiogenesis ↓ Angiogenesis ↓ Angiogenesis Biological activity 2- macrophage colony- but- methyl- 3- 9 inhib. 9 inhib. 9785 3ct CSF, granulocyte- CSF, Aspirin Granuloma: formation, angiogenesis and hypoxia Cipemastat MMP- Sb- AB0046 Doxycycline Marimastat Batimastat MMP- Bevacizumab SU5416 Pazopanib AKB- Compound Antigen presentation and priming Mtb, Mycobacterium tuberculosis hydroxy- 4- 77 71- 187 115 115 115 70 70 117 117 Ref. ; HMBPP, (E)- HMBPP, ; α Mtb Mtb Mtb Mtb Pathogen Mtb, Mm Mtb, Mm Mtb, Mm Mtb Mtb B4; matrix and MMP, metalloprotease. , tumor necrosis factor- leukotriene- (R)CT α 4, in vitro, in vivo, in vivo, (R)CT in vitro in vitro, in vivo Model in vitro, in vivo in vitro, in vivo in vitro, in vivo in vitro in vitro ; IL,; interleukin; TNF- γ protective protective protective protective protective protective protective epigenetics epigenetics epigenetics epigenetics epigenetics epigenetics epigenetics ↑ Reactive oxygen species ↑ Reactive oxygen species ↑ Host- ↑ Host- Biological activity ↑ Host- ↑ Host- ↑ Host- ↑ Host- ↑ Host- , interferon- γ

a a glucose; ATP, adenosine glucose; ATP, triphosphatases; LTB

(Continued)

a a D- deoxy- Compound Phagocytosis and phagosome maturation Metformin Celecoxib Epigenetic regulation TMP195 TMP269 Trichostatin A Resveratrol SRT1720 Valproic acid SAHA Compounds targeting distinct host intracellular pathways are categorized under multiple sections. (randomized) clinical trial; CR, case report; PRR, pathogen recognition receptor; inhib., inhibitor; GM- TABLE 1 hydroxamic acid; IFN- 2- a KILINÇ et aL | 7

Mtb, positioning PPRs as potential HDT targets to combat mycobac- Formation and subsequent acidification of phagolysosomes is terial infections.13- 17 Furthermore, also downstream PRR signaling also inhibited by Mtb- secreted protein 1- tuberculosinyladenosine might be modulated by HDT. TLR2- dependent expression of miRNA- antacid (1- TbAd).30 Accumulation of 1- TbAd in acidic intracellular 125 hampered autophagy in murine macrophages.18,19 TLR2- MyD88 compartments resulted in swelling and ultimately bursting of phago- signaling in Mtb- infected murine macrophages was also repressed somes, permitting mycobacterial escape into the cytosol. To further by upregulation of miRNA- 23a- 5p, restricting Mtb infection– induced impair phagosome integrity, Mtb permeabilizes phagosomal mem- autophagy and thus increasing intracellular Mtb survival.20 Inhibition branes using the bacterial ESX- 1 secretion system (ie, ESAT- 6),31 of miRNA- 12518,19 or miRNA- 23a20 both reduced Mtb survival, iden- which leads to leakage of phagosomal cargo into the cytosol, allowing tifying miRNA- 125 and miRNA- 23a as potential targets for HDTs. phagosomal escape of mycobacteria. Although the cytosol contains After phagocytosis, mycobacteria become localized in phago- an abundance of nutrients to support bacterial growth, translocation somes that are initially non- degradative, but slowly mature into into the cytosol also activates DNA- and RNA- sensing pathways via increasingly hostile organelles. This so- called phagosome matura- intracellular recognition of PAMPs and danger- associated molecular tion hinges upon fusion with lysosomes that contain antimicrobial patterns (DAMPs) to induce antimycobacterial host effector mech- peptides and induce intravesicular acidification enhancing lysosomal anisms. Retinoid acid– inducible gene I (RIG- I)– like receptors are cy- enzyme activity.21 While initially thought to be simply transport tosolic PRRs recognizing single- and double- stranded RNA and upon vehicles, phagosomes have appeared to be highly dynamic struc- ligation induce the type- I IFN pathway, among others.32 Nucleotide- tures that are regulated by several membrane markers, such as PI3P, binding oligomerization domain (NOD)– like receptors are intracellu- acidifying proton adenosine triphosphatases (ATPases), and Rab- lar sensors for several DAMPs and PAMPs, including bacterial RNA, GTPases.21,22 As GTPases are also involved in autophagy induction, that can induce both type- I IFN and IL- 1 responses.32 Enhancing ex- these enzymes could be interesting targets for HDT, but as of yet pression levels of RIG- I- like receptors using nitazoxanide treatment have not been investigated in this context. To prevent (auto)phago- during mycobacterial infection increased IFN-β levels and concomi- some maturation, Mtb secretes proteins such as SapM and PknG, tantly reduced mycobacterial loads in an in vitro TB model,33 but did which inhibit PI3P- phosphorylation, dissociation of early endosomal not show efficacy in TB patients, possibly due to negligible concen- protein Rab5 and acquisition of late endosomal protein Rab7.23 In trations at the site of infection.34 addition, Mtb prevents recruitment of the proton pumping enzyme vacuolar- type H+- ATPase (v- ATPase) by phagosomes, thus further arresting phagosome acidification.24 Several receptor tyrosine ki- 2.2 | Autophagy nases (RKTs) that are activated upon internalization of Mtb and NTM are involved in both bacterial uptake and intracellular trafficking, Autophagy is a mechanism mediating self- maintenance and cellular and could be exploited as targets for HDT.25 Abelson tyrosine ki- homeostasis and is induced under stress such as hypoxia, starvation nase (Abl), involved in bacterial uptake by regulating cytoskeletal but also microbial infection.35 Autophagy is crucial during Mtb and dynamics in host cells, can be chemically inhibited by imatinib,24 and NTM infections and inhibition of autophagy using azithromycin in- indeed, imatinib treatment impaired internalization of Mtb by human creased susceptibility of cystic fibrosis patients to NTM infection.36 macrophages.26 Furthermore, Abl also modulates the expression of Autophagy is initiated by formation of a double- membraned v- ATPase, and inhibiting RTKs with imatinib induced expression of phagophore that, under stringent control of ubiquitin- like protein several v- ATPase pump- subunits and their colocalization with Mtb- conjugation systems, expands around the intracytoplasmic cargo containing phagosomes, promoting phagosomal acidification and to form autophagosomes, which ultimately fuse with lysosomes enhancing bacterial killing in human macrophages.27 In line with to mediate degradation. Two autophagic pathways are important this, imatinib treatment of mice infected with Mtb or Mycobacterium for mycobacterial degradation: LC3- associated phagocytosis (LAP) marinum (Mm) decreased mycobacterial load and combining ima- and the STING- dependent cytosolic pathway.37 LAP is initiated by tinib with first- line anti- TB drug rifampicin synergistically reduced downstream signaling of numerous receptors, including TLRs,37 mycobacterial load in mice and in a murine macrophage- like cell after which the phagosome becomes decorated with PI3P produced line.25- 27 The potential of inhibiting host tyrosine kinases to im- by the PI3KC3 complex, that includes Beclin- 1 and Rubicon. PI3P pair intracellular mycobacterial survival is further highlighted with and Rubicon are required for the generation of reactive oxygen AZD0530 treatment, a Src- inhibitor, that lowered disease burden in species (ROS) and conjugation of lipidated LC3- II to the membrane Mtb- infected guinea pigs by promoting phagosomal acidification.28 to enhance phagosomal maturation.37 The STING- dependent path- Moreover, Korbee et al showed that inhibiting RTK signaling with way is triggered by mycobacterial DNA released into the cytosol the repurposed drugs AT9283, ENMD- 2076 and dovitinib signifi- through the bacterial ESX- 1 system. When mycobacterial DNA is cantly reduced intracellular Mtb in primary human macrophages.29 sensed by a STING- dependent DNA sensor, cytosolic Mtb is ubiq- Being repurposed, these compounds are already FDA- approved or uitinated by the ubiquitin- ligating (E3) ligase, bound to autophagic in phase II and III clinical trials, thus accelerating potential clinical receptors including p62/sequestosome 1, NDP52 protein and TBK1, application as adjunct therapy in treating (MDR)- TB and treatment and subsequently delivered to autophagosomes by engagement of of refractory NTM infections. membrane- associated LC3.32 8 | KILINÇ et aL

Numerous drugs have been identified that promote autophagy In addition to Beclin- 1 and TBK1, other components of the au- by targeting different components of the autophagic pathways. tophagic pathways have also been targeted to promote mycobac- Beclin- 1 is induced by human antimicrobial peptide (AMP) LL- 37, terial clearance. Ca2+ signaling is pivotal in inducing autophagy by also known as cathelicidin.38 Cathelicidin is able to suppress Mtb activating the Ca2+/calmodulin- dependent serine/threonine- kinase growth and can be induced by pathogens after TLR2/TLR1 ligation, (CaMKK2)/ULK1 complex.65 CaMKK2- mediated autophagy and kill- and also by vitamin- D.39 In vitro experiments identified calcitriol, the ing of intracellular Mtb requires Ca2+ transporter CACNA2D3 which bioactive metabolite of vitamin- D, to exert antimicrobial activity by is, however, suppressed by Mtb- induced miRNA27a.66 Intracellular mediating intracellular killing of Mtb through cathelicidin.40 Calcitriol survival of Mtb could be impaired by inhibiting miRNA- 27, providing has also been linked to nitric oxide (NO) production and suppression a new HDT target. of matrix metalloproteases (MMPs) which may further protect the Another important negative regulator of autophagy is the PI3K- host from TB immunopathology.41,42 The efficacy of vitamin- D as Akt- mTOR signaling pathway, which is robustly activated by Mtb to HDT during TB disease has been investigated in multiple random- facilitate its intracellular survival.67 Everolimus, an improved analog ized controlled trials (RCTs). Vitamin- D administration corrected of mTOR inhibitor rapamycin, was able to reduce Mtb burden in a any vitamin- D deficiencies and was safe in use but did not show human granuloma model and these effects were additive to first- line consistent beneficial outcomes during mycobacterial infections in TB drugs, possibly by HDT activity and/or by inhibiting mycobacterial meta- analyses.43- 45 Acceleration of Mtb clearance from sputum was growth directly.68 Inhibition of protein- kinase C- beta (PKC- B), another mainly observed in MDR- TB cases or patients with a specific gen- important regulator of the PI3K- Akt- mTOR pathway, by ibrutinib otype, such as polymorphisms in the vitamin- D receptor- gene.46,47 also enhanced autophagy and restricted intracellular growth of Mtb Furthermore, low levels of vitamin- D have been linked to a higher in macrophages and mice in the spleen, although not in the lungs.69 susceptibility to develop TB disease.48 Some studies combined Alleviating the Mtb- mediated suppression of sirtuin- 1, a class- III his- vitamin- D therapy with phenylbutyrate (PBA), which stimulates tone deacetylase that also modulates autophagy via 5’AMP- activated cathelicidin- induced autophagy and also inhibits bacterial growth protein- kinase (AMPK), using resveratrol restricted intracellular Mtb directly.49,50 Combining vitamin-D and PBA treatment further in- growth by stimulating autophagy and phagosome- lysosome fusion.70 creased expression of cathelicidin in healthy volunteers, but the aug- Metformin, a well- established stimulator of AMPK- mediated inhibi- mented expression level was constrained to a defined dose range of tion of mTOR signaling, is widely used for the treatment of type- 2 PBA.51 The narrow therapeutic window of PBA might clarify why diabetes, but also induces ROS production, phagosome maturation, certain RCTs failed to detect accelerated sputum- smear conversion and autophagy in vitro and prevents mitochondrial membrane depo- by co- administering vitamin- D and PBA52 and only showed acceler- larization.71- 73 In non- diabetic healthy volunteers, metformin treat- ated sputum- smear conversion at week 4 following combined treatment downregulated genes involved in Mtb- mediated modulation of ment, but not at week 8 in vitamin- D- deficient patients.53 Due to autophagy, as well as type- I IFN signaling, while upregulating genes these inconsistencies, progression of vitamin- D as potential HDT in involved in phagocytosis and ROS production.74 Several clinical trials TB treatment regimens has not been successful. have shown that metformin treatment reduces the risk of latent TB Vitamin- A- deficiency has also been correlated with an in- reactivation and TB mortality, and in patients with cavitary TB, im- creased risk to develop TB disease.54 STING- dependent autoph- proves sputum culture conversion.75- 77 agy can be targeted by the active metabolite of vitamin- A, that Like metformin, repurposing of drugs that are clinically approved is, all- trans retinoic acid (ATRA), which promotes TBK1- mediated in the context of other diseases has been shown to enhance auto- enhancement of autophagy which reduces Mtb survival in human phagy and to reduce intracellular bacterial growth, suggesting these macrophages.55 ATRA is also known to increase CD1d receptor drugs may be considered as HDT candidates. The anti- cancer drug expression on innate immune cells,56 and treatment with non- gefitinib, an inhibitor of epidermal growth factor receptor (EGFR), mycobacterial CD1d ligand α- galactosylceramide (α- GalCer) re- promotes intracellular Mtb killing by alleviating the STAT3- dependent duced mycobacterial load and improved survival of mice with repression of effective immune responses in Mtb- infected mice and 57 TB, and while α- GalCer combined with ATRA and vitamin- D did by enhancing lysosomal biogenesis and targeting of mycobacteria not clear the infection in mice, it improved containment of the in- to lysosomes in Mtb- infected macrophages.78 Gefitinib also induced fection.58 In patients, vitamin- A supplementation combined with autophagy,78 but since no specific targeting of mycobacteria to the Zn2+ or vitamin- D gave inconsistent results.59- 62 Thus, although autophagic pathway was observed, this activity has not been for- vitamin- A reduced Mtb loads in vitro and in vivo, evidence for its mally linked to restricting intracellular Mtb survival. Bazedoxifene, efficacy in patients is inconsistent. An additional regulator of the a selective estrogen receptor modulator (SERM) used for breast selective STING- dependent autophagy pathway is DNA- damage cancer treatment, was also shown to inhibit intracellular Mtb growth regulated autophagy- modulator protein 1 (DRAM1). DRAM1 was in macrophages through enhanced ROS- dependent autophagy79 found to trigger autophagy in both Mtb- infected human mac- and to inhibit Mtb growth in liquid culture. Furthermore, one study rophages and Mm- infected zebrafish larvae, whereas DRAM1- showed that loperamide, an anti- diarrheal drug, promoted autoph- deficiency resulted in host- detrimental cell death, underscoring agy as indicated by p62 degradation and decreased mycobacterial DRAM1 as an interesting target for HDT.63,64 burden in vitro and ex vivo in murine macrophages.80 KILINÇ et aL | 9

Mtb not only inhibits the initiation of autophagy, but also fusion Although ROS production is important for host resistance of autophagosomes with lysosomes via protein P2- PGRS47.23,81 against mycobacterial infection, modulating ROS as HDT strat- Furthermore, the Mtb secretion- factor SapM inhibits Rab7- egy requires careful monitoring as excess ROS leads to oxidative recruitment to prevent autophagosome- lysosome fusion.82 Mtb- stress and concomitant necrosis.98 Corroborating this view, re- expressed mannosylated lipoarabinomannan (ManLAM) also inhibits ducing ROS accumulation in Mtb- infected macrophages with ROS maturation of autophagosomes, by blocking LC3- translocation to scavenger N- acetylcysteine in fact restricted Mtb replication and autophagosome membranes.32,83 Releasing such blockades in restored macrophage cell viability,99 and in a guinea pig Mtb infec- autophagosome- lysosome fusion could represent potential HDT tion model N- acetylcysteine administration was also shown to be strategies. Bedaquiline, a novel antibiotic now in use for MDR- TB, efficacious.100 Moreover, N- acetylcysteine was found to be safe in has also been shown to induce phagosome- lysosome fusion and au- a cohort of TB- HIV coinfected individuals,101 although its impact tophagy via activation of TFEB, possibly contributing to it successful on culture conversion remains to be determined.101 Nevertheless, application as a new TB drug.84 In line with this, a small molecule ROS production is important for the bactericidal activity of macro- called 2062 improved autophagy and lysosomal pathway activity phages102 and the critical balance in ROS production and its regu- via activation of TFEB when administered with suboptimal doses of lation is important in restricting intracellular mycobacterial growth rifampicin.85 without harming the host. Although autophagy- targeting HDTs have been investigated Multiple studies in mice and humans have shown antimicrobial mainly in the context of Mtb infections, several case reports have effects of NO, but the exact underlying mechanisms remain un- been published for (disseminated) Mav infections in patients who clear.103,104 Macrophages from LTBI patients were shown to control received granulocyte- macrophage colony- stimulating factor (GM- Mtb growth via NO production, and human macrophages required 105 CSF) or IFN-γ . GM- CSF treatment during Mtb infection reduced iNOS for intracellular killing of Mtb. Moreover, compared to wild- bacterial burden by promoting phagosome- lysosome fusion and type murine macrophages, protein- kinase R (PKR)– deficient macro- 106 increased expression of TNF-α , IFN-γ , and inducible nitric oxide phages induced higher levels of iNOS during Mtb infection, and synthase (iNOS).86- 88 GM- CSF treatment during Mav infection PKR- deficient mice had lower Mtb loads and less severe lung pathol- increased phagocytosis and impaired bacterial growth in vitro in ogy compared to infected wildtype mice, highlighting the potential human macrophages and in Mav- infected patients with or with- of PKR as HDT target. Despite its importance as substrate for NO out HIV infection (partially) improved clinical outcome.89- 92 Thus, production, supplementing L- arginine did not consistently improve autophagy likely plays an important role also in NTM immunity, clinical outcomes such as cure rate or (sputum) smear conversion in and could represent an attractive target for HDT in severe NTM several clinical trials.107- 109 infections. Several Mtb- associated proteins have been identified that protect Mtb from RNS, but Mav naturally tolerates intracellular NO levels and may even benefit from host NO.110- 112 Mice that cannot 2.3 | Intracellular killing mechanisms: reactive produce NO were more resistant to Mav infections, while being more oxygen and nitrogen species prone to Mtb infections.113 In agreement with this, compared to wildtype mice, NOS2- deficient mice showed higher IFN-γ responses To eliminate mycobacteria during infection, host cells trigger the during Mav infection and increased accumulation of especially CD4+ production of reactive oxygen species (ROS) and reactive nitrogen T- cells.114 Enhancing NO production can thus be beneficial in com- species (RNS), via NADPH oxidase 2 (NOX2)93 and inducible nitric batting mycobacterial infections such as Mtb, but not Mav. oxide synthase 2 (iNOS), respectively. iNOS catalyzes production of nitric oxide (NO) by converting L- arginine into L- citrulline, which is subsequently converted into RNS.94 Once expressed, both ROS 2.4 | Epigenetic regulation and RNS interact with the phagosome to destroy bacterial components.95 Mycobacterium- induced ROS production occurs via the Macrophage polarization is an important mechanism of the immune TLR(2)- MyD88 signaling axis and impairments in this pathway in- system to respond adequately to the plethora of pathogens, which crease susceptibility to Mtb infection.7,96 Recently, TLR2- dependent is partly mediated by epigenetic regulation of gene expression using ROS production and bactericidal activity was found to be impaired histone acetylation. The level of histone acetylation is regulated by in CD157- deficient murine macrophages which could be rescued by the balanced activity of histone acetyltransferases (HATs) and his- administration of soluble CD157.95,97 Moreover, expression levels of tone deacetylases (HDACs). HDACs are divided into four classes, CD157, an enzyme important for leukocyte migration and involved three of which are Zn2+- dependent (class I, IIa/IIb and IV), while in nicotinamide- adenine- dinucleotide (NAD+) metabolism, are ele- class-III is NAD+- dependent.115 Mtb infection actively modulates vated in patients with active TB compared to LTBI and lowered when the acetylation status of host histones by (a) suppressing expres- patients are treated with TB chemotherapy, indicating an important sion of class- II HDACs (ie, HDAC 3, 5, 7, and 10) in macrophages, role of CD157 in host immunity, biomarker profiling and also provid- with anti- inflammatory M2 macrophages being more affected than ing a potential HDT.97 pro- inflammatory M1 macrophages,115 (b) inhibiting the expression 10 | KILINÇ et aL of class- III HDAC sirtuin- 1 both in vitro and in human tissues from MHC-class II. By actively engaging TLR2 rather than other TLRs, Mtb TB patients,70 and (c) upregulating expression of sirtuin- 2, another (and to a lesser extent M smegmatis) minimizes upregulation of CIITA class- III HDAC that regulates cell cycle and metabolism.116 Type IIa– and concomitant MHC- class II expression. In addition, TLR2 (among specific HDAC inhibitors (HDACi) TMP195 or TMP269 reduced bac- all TLRs) most potently induces an innate (IL- 6) response,120 leading terial loads in M2, but not M1 macrophages, while broad- spectrum to upregulation of suppressor- of- cytokine- signaling- 1 (SOCS1) that HDACi trichostatin A reduced bacterial loads in both M1 and M2 in turn inhibits signal- transducer- and- activator- of- transcription 1 macrophages. Interestingly, combining HDACi with AKT1 kinase (STAT1) phosphorylation and antigen presentation, further impair- inhibitor H- 89 resulted in cumulative reduction in bacterial loads. ing the adaptive host immune response.23 MiR106b, which degrades Importantly, in a Mm zebrafish infection model, both class- IIa and mRNA encoding cathepsin S, a protein that modulates MHC- class II pan- HDAC inhibition reduced bacterial loads, confirming the in molecules to allow peptide loading, is significantly upregulated dur- vivo potential of HDAC inhibition as HDT to treat TB.115 Sirtuin- 1, ing Mtb infection.121 Inhibition of miR106b using miRIDIAN hairpin a class- III HDAC important during (viral) infections, regulates stress inhibitors upregulated expression of both cathepsin S and HLA-DR responses and cellular metabolism. Resveratrol or SRT1720, a and enhanced subsequent CD4 T- cell proliferation.121 Alternatively, natural and synthetic activator of sirtuin- 1, enhanced clearance of inhibition of sirtuin- 2 activity in macrophages using AGK2 modu- drug- sensitive and drug- resistant Mtb.70 Both compounds stimulate lated gene expression- promoted antigen presentation.116 AGK2 autophagy and phagolysosome fusion in THP- 1 cells, which likely ac- treatment of mice resulted in upregulation of MHC- class II expres- counts for the enhanced bacterial killing, while reducing pathology sion but also of costimulatory molecules and markers of activation, 116 in a TB mouse model, possibly by inhibiting expression of IL- 1β, IL- 6, leading to enhanced priming of T- cells and improved Mtb killing. 70 MCP- 1, and TNF-α . Since Mtb limits activation of antigen- presenting cells (APCs), The Mtb genome encodes Rv1151c, a sirtuin- like NAD- which precludes the host from mounting an effective adaptive im- dependent deacetylase, allowing Mtb to produce acetyl coenzyme mune response, proper activation of APCs could be an interesting A (acetyl- CoA) synthetase, a critical enzyme in energy metabo- HDT. A possible strategy for HDT could be administration of G1- lism of both host cells and bacteria. Targeting this pathway using 4A, a polysaccharide from Tinospora cordifolia that presumably sig- HDACi valproic acid directly inhibited bacterial growth, likely by nals via TLR4, or TLR4 ligand LPS combined with a CD40 agonistic inhibiting acetyl- CoA production by Mtb itself, while co- treatment antibody.13,122 Both treatments induced vast cytokine production of valproic acid and rifampicin/isoniazid therapy resulted in cumu- (IFN-γ /IL- 12, TNF-α , IL- 6) and upregulation of costimulatory mol- lative effects.117 By contrast, FDA- approved HDACi suberoylanilide ecules by dendritic cells in vitro.122 Furthermore, both treatments hydroxamic acid (SAHA) had no direct effect on Mtb growth, but reduced bacterial loads in murine TB infection models which was, at it reduced mycobacterial growth via host- directed mechanisms and least in part, T- cell- mediated.122 However, systemic administration synergized with rifampicin/isoniazid therapy.117 As Rv1151c is well of TLR ligands is known to cause significant side effects,123 and may conserved across different mycobacterial species including Mav,118 only be applicable via local administration. Bergenin, a phytochemi- the above therapies may also be efficacious against NTM. cal extracted from tender leaves, enhanced macrophage activation, as evidenced by enhanced CD11b expression as well as augmented NO, TNF-α , and IL- 12 production, through activating the MAPK/ 3 | HDT MODULATING ADAPTIVE IMMUNE ERK pathway. The resulting increased IL- 12 production induced a RESPONSES robust Th1 response with concomitant IFN-γ production by T- cells. Bergenin therapy reduced bacterial loads as well as lung pathology 3.1 | Antigen presentation and priming in a murine TB infection model.124 Of note, vaccination could also be an interesting HDT approach to activate APCs or reprogram an Upon phagocytosis, pathogens are processed and degraded, such effective adaptive immune response. This, however, falls outside the that pathogen- derived peptides can be loaded and presented in scope of this review and is excellently reviewed elsewhere.125 MHC- class I and II molecules to initiate adaptive T- cell responses. Due to the chronic immune stimulation during persisting myco- One strategy of mycobacteria to evade host adaptive immune re- bacterial infections, including LTBI, T- cells and APCs upregulate inhib- sponses is to impair presentation of mycobacterial peptides by evad- itory receptors such as PD- 1/PD- L1, which can impair T- cell effector ing phagosomal degradation. Improving mycobacterial degradation functions,23 and may be interesting targets for HDT. Expression by promoting phagosomal maturation and/or autophagy induction of exhaustion- associated markers by T- cells during active disease, as discussed above likely enhances both antigen presentation and however, is rather ambiguous: Despite successes in anti- cancer ther- concomitant adaptive immunity. Another strategy of mycobacte- apies by inhibiting immune checkpoint molecules with anti- PD- 1/ ria to evade host adaptive immune responses is to predominantly PD- L1 antibodies, PD1/PDL1- directed experimental therapies in in infect macrophages instead of dendritic cells, the former requiring vitro and in vivo TB models resulted in impaired intracellular control stronger activation before being able to efficiently process and pre- of Mtb and TB exacerbation rather than improved resolution,126,127 sent antigens for priming naive T- cells.119 Macrophage activation is suggesting PD- 1 may be a T- cell activation rather than exhaustion required to induce expression of CIITA, a major positive regulator of marker during TB. Moreover, reports of LTBI- reactivation in cancer KILINÇ et aL | 11 patients treated with anti- PD- 1/PD- L1128 warrant a cautionary note hallmark Th2- cytokine, impaired mycobacterial control by human against this therapy in TB. macrophages and enhanced the proportion of regulatory T- cells in Indoleamine 2,3- dioxygenase (IDO) expression is actively in- vitro.131 Blocking IL- 4 completely alleviated these effects and im- duced by mycobacteria in animal (macaques and mice) models of proved bacterial control,131 suggesting Th skewing could be an inter- acute TB, but not LTBI, and IDO levels correlated with bacterial esting target for HDT. burden. IDO catabolizes tryptophan into kynurenine, which in Alternatively, administration of IL- 2, which stimulates T- cell pro- turn suppresses IFN-γ production by CD4 T- cells, a cytokine piv- liferation while inhibiting T- cell anergy, in patients infected with otal in the anti- TB response, identifying IDO as potential target drug- resistant Mtb has been investigated in five RCTs and compared for HDT. In vivo inhibition of IDO activity using D- 1MT one week in a meta- analysis.146 While CD4 T- cell numbers increased and time after mycobacterial infection enhanced T- cell proliferation and to culture conversion improved, radiographic changes were not ob- differentiation in effector and memory cells while apoptosis was served compared to standard chemotherapy.146 In mice infected enhanced.129 Furthermore, D- 1MT treatment improved penetra- with Mav, IL- 2 therapy resulted in decreased bacterial burden,147 tion of T- cells into granulomas, likely allowing protective T- cell- whereas mixed results were described in case reports.148,149 The lim- mediated granuloma reorganization, and reduced bacterial loads ited effect of IL- 2 therapy may be due to immune suppression caused and lung pathology.129 by expansion of regulatory T- cells and myeloid- derived suppressor cells (MDSC), both expressing elevated levels of the high affinity IL- 2 receptor, as depletion of these suppressor cells improved outcome 3.2 | Skewing of T- cells in a mouse TB model.150 Combining IL- 2 therapy with mycobacterial phosphoantigen (E)- 4- hydroxy- 3- methyl- but- 2- enyl pyrophosphate Th1- responses, characterized by high IFN-γ secretion, are cru- (HMBPP) in nonhuman primates induced significant expansion of 130- 132 cial in effective anti- Mtb immune responses. Nevertheless, Vγ2Vδ2 T- cells that migrated to the lungs, evoking a Th1- response Mav and Mtb reduce cellular responses to IFN-γ and deficiencies that significantly reduced mycobacterial burden as well as lung pa- 151 in the IL- 12/IL- 23/IFN-γ - axis increase susceptibility to Mav infec- thology. Rather than systemic administration of cytokines, which tions.133,134 In several patients suffering from pulmonary TB, direct frequently results in systemic side effects, ex vivo stimulation of au- administration of IFN-γ accelerated sputum- smear conversion and tologous peripheral blood mononuclear cells (PBMCs) with a cocktail 135,136 improved chest radiograph. Administration of IFN-γ also re- of IFN-γ , IL- 2, IL- 1α, and anti- CD3 before reinfusion yielded positive duced Mav growth in murine macrophages,137 and improved clini- results with minimal side effects in a case report with MDR- TB.152 cal outcome (ie, decreased respiratory symptoms and mortality) in This, however, requires further clinical investigation. several but not all Mav- infected individuals,138- 140 suggesting potential of IFN-γ as HDT in both Mtb and Mav infections. In vivo administration of IL- 12, a key cytokine that drives Th1 skewing, en- 4 | (PROGRAMMED) CELL DEATH hanced IFN-γ and TNF-α responses and significantly reduced bacterial burden in an acute mouse TB model.141 Similarly, restoring Several types of cell death can follow mycobacterial infection of IL- 24 expression in a mouse TB model enhanced Th1- responses and macrophages: apoptosis, necrosis, and ferroptosis.32 During apop- IFN-γ production, with concomitant improved survival and reduced tosis, bacteria remain encapsulated, which facilitates bacterial clear- bacterial loads.142 A large proportion of human Mtb- specific CD4 ance; however, pathogenic mycobacteria have developed strategies 153 Th1- cells expresses CCR6 and co- produces IFN-γ /IL- 17, often de- to limit apoptosis. Activation of transcriptional regulator peroxi- picted as Th1* or Th1- 17 cells, and being associated with LTBI sug- some proliferator- activated receptor gamma (PPARγ) by ManLAM, gest their importance in protection against active TB.143 However, stimulating mannose receptors, upregulated (pro- host- cell survival) IL- 17 responses during TB need to be carefully regulated to prevent Mcl- 1 and repressed (pro- apoptotic) Bax without Bak and improved 154,155 neutrophil- driven lung pathology, which is mediated by regulatory host- cell survival. In agreement with the PPARγ- dependent in- T- cells as well as so- called regulatory CD4 Th17- cells that co- hibition of host- cell apoptosis and concomitant antimycobacterial produce IL- 17 and IL- 10.144 In case of disbalanced Th17 responses immunity, direct pharmacological inhibition of Mcl- 1 resulted in re- with concomitant excessive neutrophil recruitment, RAGE recep- duced intracellular Mtb growth in human macrophages.155 tor inhibition may be an interesting HDT. RAGE receptor is up- Besides inhibiting apoptosis, virulent Mtb stimulates host- cell ne- regulated during active TB disease and after ligation with S100A8/ crosis, which allows infection to disseminate to neighboring cells.32 A9 mediates neutrophil recruitment.145 In a TB model, mice defi- Mtb can induce necrosis via the virulence factor tuberculosis necro- cient in S100A8/A9 had reduced bacterial loads, neutrophil influx tizing toxin (TNT), which is secreted into the cytosol where its NAD+ and pathology compared to wildtype. Moreover, inhibition of the glycohydrolase activity depletes the host cell from NAD+,156 leading RAGE receptor using FPS- ZM1 improved outcome comparably as to permeabilization of mitochondrial membranes, decreasing ATP- S100A8/A9- deficiency.145 production and activating necrosis. Nicotinamide- based HDT allevi- Th2- responses have been associated with active cavitary TB ated necrosis- induced host- cell cytotoxicity in Mtb- infected cells by disease or TB treatment failure,130,131 and administration of IL- 4, a replenishing NAD+.99 Mtb can furthermore induce necrosis mediated by 12 | KILINÇ et aL mitochondrial membrane permeability transition via p38- MAPK phos- a result of ATP depletion induced macrophage apoptosis.174 An impor- phorylation, which can be inhibited by corticosteroids dexamethasone tant enzyme during glycolysis, lactate dehydrogenase (LDH), which and doramapimod.157 In addition, corticosteroids dexamethasone and converts pyruvate into lactate, is significantly upregulated during Mtb prednisolone, both well- known general immunosuppressants, have infection.175 Although the pathophysiology of LDH upregulation re- also been investigated as HDT during mycobacterial infections. With mains to be addressed, pharmacological inhibition of LDH using FX11 some reports of improved survival,158,159 likely by limiting secretion reduced bacterial load and development of necrotic lesions in granu- of pro- inflammatory cytokines, meta- analysis failed to show a signif- lomas in a murine TB model, suggesting a significant role of LDH in icant improvement in clinical outcome after corticosteroid therapy in driving disease and a potential target for HDT.175 Interestingly, while patients with TB.160 Interestingly, while promoting pro- inflammatory ATP depletion can induce macrophage apoptosis (considered host pro- cytokine levels of TNF-α by adenylate cyclase inhibitor (SQ22536) or tective), exogenous ATP activates macrophages via the P2RX7/P2X7 a PKA inhibitor (H- 89) has been shown to improve control of infection receptor and also directly inhibits growth of mycobacteria, including 161 176,177 by stimulating mitochondrial ROS production, excess TNF-α leads Mtb and Mav, due to chelation of iron. ATP treatment has already to membrane disruption and ATP depletion via mitochondrial enzyme been shown to synergize with standard Mav antibiotic treatment, mak- cyclophilin D, which together with lysosomal enzyme acid sphingo- ing ATP an interesting adjunctive HDT molecule to enhance chemo- myelinase induced necrosis.162,163 Alisporivir and desipramine, two therapeutic efficacy against mycobacterial infections.177 In addition, clinically approved drugs that inhibit cyclophilin D and acid sphingomy- an FDA- approved potentiator of P2RX7/P2X7, clemastine enhanced 178 elinase, respectively, prevented TNF-α - induced necrosis without com- mycobacterial killing in a zebrafish model. This may provide a poten- 163 promising TNF-α - induced ROS- dependent mycobacterial killing. tially attractive avenue to explore synergistic effects between ATP and Correspondingly, upregulation of cAMP levels by phosphodiesterase clemastine treatment in future studies. (PDE) inhibitors cilostazol and sildenafil decreased TNF-α levels, result- Conversion of pyruvate into acetyl coenzyme A (Ac- CoA) ini- ing in reduced immunopathology and fastened bacterial clearance in tiates the tricarboxylic acid (TCA) cycle which produces energy 164,165 Mtb- infected mice. Blocking TNF- α, which facilitates necrotizing using OXPHOS. During Mtb infection, several enzymes important granulomas during active TB, displayed promising results in preclinical in the TCA cycle are downregulated and TCA cycle intermediates, 166,167 animal models. However, blocking TNF-α also leads to disease such as citrate, are translocated from mitochondria into the cyto- reactivation and concomitant dissemination in LTBI patients and in the sol. Typically, citrate is converted into itaconate which dampens absence of standard TB chemotherapy exacerbated disease severi- tissue hyperinflammation by suppressing both ROS production and 168- 170 ty, precluding clinical application of TNF-α inhibition as HDT in production of pro- inflammatory cytokines such as IL- 1β, IL- 6, and IL- 174 TB. The balance between TNF-α - mediated beneficial and detrimental 12. In the cytosol, however, citrate is cleaved into Ac- CoA, which effects on host control of TB and likely other mycobacterial infections is either converted into arachidonic acid or into mevalonate and including NTM is thus delicate. Taken together, these data indicate that malonyl- CoA. This leads to synthesis of eicosanoids, cholesterol, Mtb- induced host- cell necrosis favors mycobacterial survival and this and free fatty acids, respectively, of which the latter two are stored can be effectively counteracted by HDT, while the double- edge sword intracellularly in lipid droplets.174 Hypercholesterolemia results in of modulating TNF-α levels currently prohibits clinical application. spontaneous formation of lipid droplets in macrophages. Further Ferroptosis is a type of necrosis characterized by accumulation accumulation of intracellular lipid droplets is actively stimulated by 171 174,179 of free iron and toxic lipid peroxides. In Mtb- infected cells expres- Mtb and enhanced by both IL- 6 and TNF-α signaling, while IL- 174 sion of glutathione peroxidase- 4 (GPX4) is reduced, leading to failure 17 and IFN-γ limit intracellular lipid accumulation. Lipid- loaded of glutathione- dependent antioxidant defenses and cell death.172 macrophages are impaired in killing intracellular mycobacteria (ie, Inhibiting ferroptosis by ferrostatin 1 reduced bacterial burden both Mtb, Mav, and BCG)180 and ultimately transform into foamy macro- in vitro in human macrophages and in vivo in Mtb- infected mice.171 phages, which are associated with necrotic granulomas and tissue Furthermore, ferroptosis could also be inhibited by increasing GPX4 pathology.174 The impaired functionality of lipid- loaded macro- levels with selenium, a protein involved in GPX4 catalysis,173 show- phages involves mitochondrial dysfunction and could be restored ing that targeting this host pathway is a potential HDT strategy. using small molecule mitochondrial fusion promoter M1, which also restored macrophage bactericidal activity.180 In addition, ezetimibe, a cholesterol absorption inhibitor, prevented intracellular 5 | METABOLISM lipid accumulation and concomitantly reduced intracellular growth in Mtb- infected macrophages.179 The effects of standard antibiotic 5.1 | Carbohydrate and lipids treatment improved and perhaps even synergized with ezetimibe treatment,179 and investigating the in vivo efficacy of ezetimibe as Mtb has developed numerous strategies to modulate host metabolic well as M1 could be promising. pathways, which are broadly divided into glycolysis, oxidative phos- Statins, currently clinically used to reduce cholesterol levels, phorylation (OXPHOS), and lipid metabolism. Glycolysis conditions an could be interesting drugs to prevent lipid accumulation in macro- environment favoring Mtb growth, and inhibition of glycolysis using phages. Comparing eight different statins, simvastatin, pravastatin 2- deoxy- D- glucose (2- DG) reduced Mtb viability in one study, and as and fluvastatin were most efficacious in enhancing mycobacterial KILINÇ et aL | 13

181 killing without affecting cell viability in vitro. Mechanistically, mycobacterial infection due to absence of LTB4. Treatment with ce- while (simva)statin inhibits phagosomal acidification and degrada- lecoxib, a COX inhibitor that prevents PGE2 production and thereby 181 tion, cholesterol incorporation in (auto- )phagosomal membranes stimulates LTB4 production, or directly supplementing LTB4, re- is prevented. The presence of cholesterol in phagosomal membranes stored mycobacterial control.187 Furthermore, COX inhibitors ibu- facilitates prolonged survival of Mtb and Mav within host cells due profen and aspirin administered as single therapy or combined with to blockage of phagolysosome fusion by mechanisms not fully un- conventional TB antibiotics were shown to limit bacterial burden in derstood.182- 184 Preventing phagosomal escape ultimately enhances Mtb- infected mice,192- 194 and low- dose aspirin treatment also re- delivery of mycobacteria to (auto- )phagolysosomes and thereby duced bacterial loads in a Mm zebrafish infection model.195 Aspirin bacterial degradation.185,186 In vivo treatment with either pravasta- treatment of TB or TB meningitis patients improved survival,196,197 tin or simvastatin in a mouse TB model reduced mycobacterial loads but may impair conventional treatment regimens by reducing effi- both as a single therapy181,185 or combined with standard antibiotic cacy of isoniazid,198 but not pyrazinamide.193 Both ibuprofen and treatment.181,184 aspirin are currently tested in clinical trials as adjunct therapy for treating (drug- resistant) TB.190

5.2 | Eicosanoids 5.3 | GRANULOMA: FORMATION, Eicosanoids are lipid mediators involved in regulating inflammatory ANGIOGENESIS and HYPOXIA responses and are categorized into prostaglandins (PG), leukotrienes (LT), thromboxanes, lipoxins, and hydroxy eicosatetraenoic acids, One hallmark of TB is the extensive formation of granulomas. all of which are produced from arachidonic acid by a competing Granulomas are highly heterogenous and dynamic structures which network of enzymes, including cyclooxygenases (COX) and lipoxy- differ significantly in the level of hypoxia and available nutrients. genases.187,188 During Mtb infection, the expression of eicosanoids Granuloma formation is actively initiated by Mtb to stimulate ma- is significantly altered, with prostaglandin- E2 (PGE2) and leukot- trix metalloprotease 9 (MMP9) production. Granulomas are also 187 199,200 201 riene- B4 (LTB4) mostly upregulated. Being an immune suppres- induced during NTM infections, including Mav and Mm. sor and immune stimulator, respectively, the balance between these During initial granuloma formation non- activated macrophages are eicosanoids is highly important in regulating immunity to clear the recruited to the site of infection and serve as feeder cells for the infection, without causing tissue pathology due to excessive in- granuloma.25,202 In addition to MMP9, upregulation of several other flammation. Important in this regulation are IL- 1β and type- I IFN MMPs has been observed in lung samples from individuals infected signaling. IL- 1β signaling stimulates production of prostaglandin- with Mtb, and other mycobacteria including Mav, which may suggest E2, which is necessary to dampen the inflammation mediated by that similar mechanisms are involved.202- 206 MMPs are enzymes that pro- inflammatory leukotrienes A4 and B4 (LTA4 is the precursor of degrade and modulate extracellular matrix and are therefore key in 204 LTB 4) that are induced upon type- I IFN signaling. In severe TB, the the development of granulomas. Their expression and activity 2+ PGE2/LTA4 ratio is reduced, suggesting potential benefit of enhanc- has multiple layers of regulation. Many MMPs require Zn for their ing PGE2 signaling. Indeed, both increasing PGE2 levels using ad- activation, potent MMPs require activation by other MMPs, and ministration of exogenous PGE2 or reducing LTA4/LTB4 production their activation is inhibited by tissue inhibitors of metalloproteinases with zileuton improved host survival, while reducing bacterial loads (TIMPs). Expression of MMPs is stimulated by pro- inflammatory 189 and necrotic lung pathology in Mtb- infected mice. Moreover, cytokines, including IFN-γ , TNF-α , IL- 12, and IL- 17 and because en- combinatory therapy of zileuton with PGE2 further restricted Mtb hanced MMP- activity is associated with extensive tissue damage replication.190 during TB,203 MMPs are promising targets for HDTs. A single nucleotide polymorphism in the promotor of the gene MMP1, a collagenase that degrades collagen in the extracellular encoding LTA4- hydrolase (rs17525495), the enzyme that converts matrix, is upregulated after TLR2- ligation and due to its high potency 207 LTA4 into LTB4, has been shown to affect expression of LTA4 hydro- may drive granuloma formation during TB. In transgenic mice ex- lase, with homozygous individuals having a high (T/T) or low (C/C) pressing human MMP1, Mtb infection promoted alveolar destruc- expression.188,191 Both homozygous genotypes have poorer survival tion and collagen breakdown in lung granulomas, identifying MMP1 compared to heterozygous individuals, showing the delicateness of as a therapeutic target to limit immunopathology.207 MMP7, which is the immune balance during mycobacterial infection.188 Depending highly expressed in the cavitary wall and hypoxic granulomas, stim- on the genotype, different treatment regimens will be required, as ulates epithelial proliferation and promotes activity of other MMPs. general immune suppression using dexamethasone favored outcome Inhibition of MMP7 and MMP1 using cipemastat, a drug originally in T/T individuals, while being detrimental in C/C individuals,188,191 registered to prevent lung fibrosis, surprisingly increased cavita- suggesting the necessity of personalized HDT- based medicine tar- tion, immunopathology and mortality in mice,208 suggesting either geting eicosanoid metabolism. Mice deficient in 5- lipoxygenase, an a protective role of MMP1 or MMP7 during TB or off- target effects enzyme that stimulates production of LTA4 and thus LTB4 (thereby of the drug. The role of MMP8 is more controversial with high in- being a model for C/C individuals), were impaired in controlling terindividual variation,203,209,210 which may relate to the presence 14 | KILINÇ et aL of neutrophils in granulomas. MMP8 is more readily detectable in host cells to stimulate angiogenesis.216 Although angiogenesis could HIV- associated TB,210 suggesting that neutrophils are recruited potentially increase host- cell viability, the net effect likely favors preferentially in settings of impaired adaptive immunity. Mice defi- bacterial replication and dissemination. Blocking angiogenesis may cient in MMP9 have less granuloma formation and reduced bacterial therefore be an interesting HDT. Indeed, inhibition of VEGF using loads,211 suggesting a prominent role of MMP9 in driving disease pa- FDA- approved bevacizumab in Mtb- infected rabbits, reduced the thology. Indeed, inhibition of MMP9 expression using morpholinos total number of vessels but improved both structurally and func- reduced granuloma formation and bacterial growth in a zebrafish tionally the remaining vessels, leading to enhanced drug targeting Mm- model.25 In agreement with this concept, treatment with Sb- 3ct, to granulomatous lesions and diminished hypoxia.217 Corroborating a specific MMP2 and MMP9 inhibitor, combined with frontline TB these findings, treatment of Mm- infected zebrafish with VEGF path- antibiotics potentiated bacterial clearance both in vitro and in vivo way inhibitors SU5416, a tyrosine kinase inhibitor, or pazopanib, a in a TB meningitis mouse model.212,213 Blocking MMP9 using mono- VEGF receptor inhibitor, reduced bacterial loads and dissemination. clonal antibody AB0046 did not affect bacterial burden, but the rate Both drugs also synergized with first- line antimycobacterial drugs of relapse was reduced in a necrotic granuloma TB mouse model, rifampicin and metronidazole, a drug that targets hypoxic bacte- by mechanisms not yet fully clarified.166 Using an in vitro model for ria.218 Inhibiting vascular leakage rather than angiogenesis may be extracellular matrix degradation, treatment with doxycycline, an equally efficacious to limit nutrient supply to mycobacteria. During FDA- approved antibiotic that non- selectively inhibits human MMPs, Mm infection, angiopoietin- 2 (ANG2) is robustly induced in granu- strongly abolished Mtb- induced matrix degradation.210 In addition, lomatous lesions. ANG2 antagonizes ANG1, which promotes vessel doxycycline reduced granuloma formation in a guinea pig model, stability while limiting angiogenesis and vascular leakage. Indeed, likely resulting from abolishing Mtb- enhanced promotor activity of AKB- 9785, a molecule that mimics functions of ANG1, reduced MMP1 and by directly inhibiting bacterial growth.214 Pan-MMP in- vascular leakage and bacterial burden in a Mm zebrafish infection hibitor marimastat (BB- 2516), a collagen- peptidomimetic binding the model.219 Thus, inhibition of angiogenesis is an interesting target for active Zn2+ site contained in many MMPs, reduced granuloma size HDT to enhance drug delivery to the site of infection and combined and bacterial burden during Mtb infection in lung tissue models.203 with other therapies is likely to be even more potent. Interestingly, treatment of Mtb- infected mice with a panel of MMP inhibitors, including marimastat, as solo therapy was not effective, while all 4 small molecules enhanced in vivo potency of frontline TB 6 | PERSONALIZED AND drugs isoniazide and rifampicin, likely by blocking MMP- mediated COMBINATORIAL HDT cleavage of collagen and by improving vascular integrity, resulting in enhanced delivery of isoniazide and rifampicin to the lungs. The Although HDT could be considered as stand- alone therapy, for ex- finding that batimastat (a pan- MMP inhibitor), Sb- 3ct (a MMP2 ample, in patients suffering from total drug- resistant TB, HDT is and MMP9 inhibitor) and MMP9 inhibitor- I yielded similar results primarily envisaged as adjunct therapy in combination with classical highlights the importance of MMP9 in driving these effects.205 antibiotics. HDT might be co- administered for a limited duration at Augmenting TIMP1 activity to inhibit activity of multiple MMPs may the initiation of the standard of care regimens to shorten treatment also be an interesting HDT target. To our knowledge, however, mod- length and reduce dosage of antibiotics to minimize side effects, ulating the activity of TIMPs has not been investigated yet in the or toward the end of treatment to boost host immunity to prevent context of HDT. potential relapse. Consequently, investigating the interactions be- Central hypoxia in granulomas may initially favor host immunity tween HDT and conventional chemotherapy is pivotal, but has only as low oxygen tension increases granulysin expression in T- cells and been reported for a limited number of HDTs. Furthermore, in case NK- cells, enhancing bacterial killing in an in vitro co- culture system of undesired interactions between TB drugs and drugs for TB co- of Mtb- specific T- cells and macrophages.215 However, due to poor morbidities (eg, between rifampicin and anti- HIV therapy or anti- 220 vasculature within granulomas and hyperactive IFN-γ or possible diabetic drugs), HDT might be used to shorten current treatment superimposed IL- 4/IL- 13 released by activated T- cells, full blown regimens or possibly partially replace components of the conven- central necrosis leads to cavity formation and concomitant bac- tional chemotherapy cocktail. In line with this, interactions between terial dissemination within the host.202,211,215 Due to the hypoxic, HDT and drugs used to treat TB comorbidities should also be inves- acidic and nutrient- poor conditions in granulomas, mycobacterial tigated thoroughly. dormancy is promoted,25 and while this effectively inhibits bac- Rather than targeting one specific aspect of the inflammatory terial replication, eradication of mycobacteria is greatly hampered response during mycobacterial infections, we hypothesize that cor- because most antibiotics only affect replicating and metabolically recting the overall immunological disbalance likely is most promising. active bacteria. Furthermore, poor vascularization hampers drug de- Type- I IFN and IL- 1β signaling, regulating levels of anti- inflammatory livery in granulomas, which is further impaired due to fibrosis and prostaglandins and pro- inflammatory leukotrienes, respectively, play scarring of lung tissue caused by the disease.25 Trehalose dimyco- an important role in regulating the immune balance during mycobac- late, a mycolic acid expressed on mycobacterial cell walls, directly terial infections.189 At the time of writing, multiple randomized con- induces vascular endothelial growth factor (VEGF) expression in trolled trials investigate targeting of (one of) these pathways by HDTs. KILINÇ et aL | 15

As some TB patients suffer from overactive type- I IFN/leukotriene mycobacteria. Targeting metabolism with HDT may also help cor- signaling while others are characterized by overactive IL- 1/prostaglan- recting the balance between prostaglandins and thromboxanes, as din activity, we postulate that in this context personalized HDT would lipid droplets and cytosolic TCA intermediates are the most import- be safest and most efficacious. However, this will increase therapeu- ant sources of eicosanoids. tic costs, which could make such therapy stratifications less attractive Irrespective of what causes defective mycobacterial clearance, and feasible in lower resourced settings. To be able to predict whether improving drug delivery to the site of infection likely benefits all patients would benefit from a certain HDT, biomarkers monitoring TB patients. Angiogenesis in granulomas is significantly impaired the (immunological) status of patients may need to be identified and and further enhances hypoxia and nutrient- limitation. Targeting developed. This, however, may not be required for all HDTs as some angiogenesis during TB by (a) inhibiting VEGF (bevacizumab217), (b) HDT may improve antimycobacterial immunity in all patients. As inhibiting VEGF- mediated signaling (SU5416 or pazopanib218), or (c) mycobacteria modulate host immunity via many different pathways, antagonizing pro- angiogenesis growth factor ANG2 (AKB- 9785219) a multi- targeted approach could be necessary to fully counteract has all been shown to enhance both drug delivery and oxygenation mycobacteria- mediated host modulation. To our knowledge, however, within granulomas in animal models of TB, and may be promising only two combinations of HDT treatments have been published; com- HDTs in combination with other therapies. Despite being most fre- bining vitamin- D with PBA did not mediate additive effects compared quently investigated in combination with antibiotics, efflux pump to solo therapy, 51- 53 likely because both compounds target the same inhibitors could also improve drug delivery of HDTs. To our knowl- pathway, while in another in vitro study combining protein- kinase A/B edge, however, this has not been investigated so far but verapamil, inhibitors H- 89 or 97i with HDAC inhibitors revealed additive effects known to enhance the efficacy of rifampicin and bedaquiline against in vitro in reducing bacterial load in primary human macrophages.115 different mycobacterial infections, both in vitro and in mice,222- 224 Modulating (auto- )phagosome maturation using receptor tyro- and also chloroquine225 and piperine 226 are interesting molecules sine kinase inhibitors including imatinib,25 AZD0530,28 and multiple for combinatorial HDT. repurposed drugs recently identified in our own group29 has been Given their central and important role in orchestrating a func- shown to improve mycobacterial clearance by human macrophages tional antimycobacterial immune response, restoring (CD4 Th1/17) in vitro. Importantly, releasing the mycobacteria- mediated arrest in T- cell immunity has been pursued in many investigations. In addi- (auto- )phagosome maturation likely benefits both patients with action to enhancing activation of antigen- presenting cells, HDTs that tive disease as well as individuals with latent infection. Above, we promote phagosomal bacterial degradation (ie, stimulating auto- have reviewed multiple HDT candidates that enhance autophagy- phagy, enhancing phagosome maturation and promoting (auto- ) mediated bacterial clearance. Which of these will be most efficacious phagolysosome fusion) are all expected to enhance presentation against mycobacteria should ideally be determined in head- to- head of bacterial- derived peptides and thereby improve adaptive immu- comparisons. Metformin, being the most frequently investigated, nity. Modulating T- cell responses to restore immunity can be medi- has already been shown to reduce TB recurrence and bacterial loads ated by vaccination or T- cell cytokine therapies. Administration of in patients,75- 77 and in addition to its effects on autophagy, also en- IL- 12141 or IL- 24142 or blocking Th2 cytokine IL- 4,131 promotes Th1 221 hances mitochondrial membrane polarization, which could fur- responses with lasting IFN-γ production that may be preferred over ther enhance its efficacy. IFN-γ administration. Which of these strategies is (most) efficacious As discussed above, host- cell death pathways are actively ex- and which patients benefit most from this therapy remains to be ploited by mycobacteria to promote their survival and dissemination addressed. and have been shown to be a potential target for HDT in multiple in While most of the evidence available for host- pathogen inter- vitro and animal studies. Active clinical modulation of (programmed) actions and HDT are from TB studies, the limited number of NTM cell death in patients, however, could lead to significant adverse experimental models investigating host modulation and/or HDT effects given the complex time- and context- dependency of this emphasizes the need and urgency to understand NTM pathogen- mechanism during mycobacterial infection. esis as well as identify potentially relevant host targets. Together, Targeting metabolic pathways has been shown to be feasible these studies will help assess the safety and efficacy of HDT, pav- and represents an attractive target for HDT. While most metabolic ing the way for the introduction of HDT against a wide range of pathways are also necessary for host- cell energy production, intra- mycobacteria. cellular lipid accumulation in lipid droplets seems to mainly benefit the intracellular survival of mycobacteria. Preventing or reducing lipid droplet formation in macrophages and concomitant impaired 7 | SEARCH STRATEGY AND SELECTION immunity can be mediated by (a) limiting oxidative phosphorylation CRITERIA by, for example, stimulating polarization of macrophages toward pro- inflammatory M1- macrophages,174 (b) improving/maintaining We searched PubMed (MEDLINE) for all relevant studies pub- mitochondrial membrane potential using small molecule M1180 or lished from 1 January 2000 until 1 October 2020. The medical NAD156 and/or (c) blocking cellular cholesterol uptake using, for subject headings used were “host directed”, “HDT”, “adjunctive”, example, ezetimibe,179 which also inhibits phagosomal escape by “immunotherapeutic” or “immunomodulation” combined with 16 | KILINÇ et aL

10. Rhoades ER, Archambault AS, Greendyke R, Hsu FF, Streeter C, “mycobacterium”, “mycobacteria”, “tuberculosis”, “nontuberculous” Byrd TF. Mycobacterium abscessus Glycopeptidolipids mask un- or “NTM”. All relevant abstracts were screened independently by derlying cell wall phosphatidyl- myo- inositol mannosides blocking two researchers. The final reference list was generated based on rel- induction of human macrophage TNF- alpha by preventing interac- evance to the topics covered in this review. Only papers published in tion with TLR2. J Immunol. 2009;183(3):1997- 2007. 11. Rahman A, Sobia P, Gupta N, Kaer LV, Das G. Mycobacterium tu- English were included. berculosis subverts the TLR- 2- MyD88 pathway to facilitate its translocation into the cytosol. PLoS One. 2014;9(1):e86886. ACKNOWLEDGEMENTS 12. Gilleron M, Nigou J, Nicolle D, Quesniaux V, Puzo G. The acylation This work was supported by grants from the Netherlands state of mycobacterial lipomannans modulates innate immunity response through toll- like receptor 2. Chem Biol. 2006;13(1):39- 47. Organisation for Scientific Research (NWO- TTW) within the Novel 13. Khan N, Pahari S, Vidyarthi A, Aqdas M, Agrewala JN. Stimulation Antibacterial Compounds and Therapies Antagonising Resistance through CD40 and TLR- 4 is an effective host directed therapy (NACTAR) program (Grant No. 16444), European Union's Horizon against Mycobacterium tuberculosis. Front Immunol. 2016;7:386. 2020 research and innovation program (SMA- TB Grant No. 847762), 14. Khan N, Pahari S, Vidyarthi A, Aqdas M, Agrewala JN. NOD- 2 and TLR- 4 signaling reinforces the efficacy of dendritic cells and and the Innovative Medicines Initiative 2 Joint Undertaking (IMI2 reduces the dose of TB drugs against Mycobacterium tuberculosis. JU) under the RespiriNTM (Grant No. 853932) and RespiriTB (Grant J nnate Immun. 2016;8(3):228- 242. No. 853903) projects within the IMI AntiMicrobial Resistance (AMR) 15. Gowthaman U, Singh V, Zeng W, et al. Promiscuous peptide of 16 Accelerator program. The funders had no role in study design, data kDa antigen linked to Pam2Cys protects against Mycobacterium tuberculosis by evoking enduring memory T- cell response. J Infect collection and analysis, decision to publish, or preparation of the Dis. 2011;204(9):1328- 1338. manuscript. 16. Chodisetti SB, Gowthaman U, Rai PK, Vidyarthi A, Khan N, Agrewala JN. Triggering through Toll- like receptor 2 limits chron- CONFLICT OF INTEREST ically stimulated T- helper type 1 cells from undergoing exhaustion. J Infect Dis. 2015;211(3):486- 496. All authors declare no competing or conflicting interests. 17. Pahari S, Negi S, Aqdas M, Arnett E, Schlesinger LS, Agrewala JN. 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